The long-term goal of this research project is the development of novel, ultrasensitive and rapid diagnostic methods for rickettsial infections during the acute phase of the disease. Rickettsiae are obligately intracellular bacteria responsible for potentially lethal diseases such as epidemic and endemic typhus, scrub typhus, and Rocky Mountain spotted fever. In addition, Rickettsia prowazekii and R. rickettsii are part of the Centers for Disease Control and Prevention (CDC) and NIH category B agents and the North Atlantic Treaty Organization (NATO) select agent list for their potential use as bioterrorist/biowarfare agents (BWA). Bioterrorist attacks can occur in multiple settings, and it is widely accepted that most terrorist attacks are covert and therefore the infectious agent will be unknown until the first person becomes acutely ill and seeks medical help. A fast and accurate diagnosis is most imperative at this stage so that proper identification of the agent is performed and adequate treatment is started immediately. Current diagnostic techniques are either insensitive during the acute stage of the disease, or the techniques needed to perform the tests are cumbersome and expensive. The specific aims designed to test the hypotheses that diagnosis of rickettsial infections during the acute phase of the disease is possible by using ultrasensitive antigen detection methods and by using host biosignature analysis are: 1) Develop a highly sensitive and specific diagnostic test for rickettsial infections based on antigen detection in blood by using ultrasensitive detection methods (electrochemiluminescence and tyramide signal amplification coupled to enzyme-labeled fluorescence); and 2) Develop reliable biosignature patterns in human hosts that allow proper identification of rickettsial infections during the acute stage based on analysis of several serum analytes (proteins, hormones, and others). The research design includes production of polyclonal, monoclonal and recombinant antibodies for the development of ruthenium- and alkaline phosphatase-based sandwich immunoassays on magnetic beads to capture rickettsial organisms. The detection systems will use electrochemiluminescence and fluorescence analyzers. In addition, biosignature analysis will include 80 serum analytes and will be performed at the University of New Mexico Small Animal Core followed by computerized analysis at the Biosignature Core.